Synthesis of alkaloid pulmonarin B and its application in controlling plant viral and bacterial diseases

Abstract

The invention relates to the efficient synthesis of alkaloid pulmonarin B and its application in preventing and treating plant virus and pathogenic bacteria diseases. Based on systematic literature research and reference to the synthesis method reported by the Johan research group, the invention synthesizes pulmonarin B in 5 steps with a total yield of 59%, improves the reaction conditions, and increases the reaction yield, laying a foundation for the study of the biological activity of this type of alkaloid. The alkaloid pulmonarin B of the invention has good anti-plant virus and pathogenic bacteria activity.

Description

Technical Field

[0001] The invention relates to a highly efficient synthesis method of an alkaloid pulmonarin B and application of the alkaloid in preventing and treating plant viruses and pathogenic bacteria diseases, belonging to the technical field of agricultural protection. Background Art

[0002] Globally, plant pathogens and pests cause up to 40% of corn, potato, rice, soybean and wheat yield losses (Nat. Eco. & Evo. 2019, 3, 430-439). Plant diseases caused by bacteria, fungi, nematodes and viruses cost the global economy $220 billion each year (Front. Plant sci. 2014, 5, 660). Plant viruses account for almost half of plant pathogens, causing losses of up to $30 billion worldwide each year (Front. Plant sci. 2014, 5, 660). Because viruses are absolutely parasitic in plant cells, the substances, energy and locations required for their replication are completely dependent on the host, and plants do not have a complete and advanced immune metabolic system like animals (Science., 2014, 344(6181): 299-303), making the prevention and control of plant viral diseases particularly difficult, and they are known as "plant cancers."

[0003] Representative registered pesticides currently in use include: amino oligosaccharides (natural oligosaccharide products made of D-glucosamine linked by β-1.4 glycosidic bonds), Ningnanmycin, Ribavirin, Virus A (morpholinoguanidine hydrochloride copper acetate), etc. There are relatively few practical varieties, and the reported pesticides have a control effect of less than 60% when actually used in the field. Therefore, the development of new and efficient anti-plant virus agents is imminent.

[0004] Bio-source natural pesticides mainly refer to pesticides developed from secondary metabolites with agricultural biological activity produced by plants, animals, microorganisms, etc., such as 2.5% rotenone emulsifiable concentrate, 15% jinggangmycin water-soluble powder, etc. Discovering novel and environmentally safe pesticides from natural products is an important and efficient way.

[0005]

[0006] Pulmonarin B (Structural Formula 1) is a natural product with an aromatic amide structure. In 2014, Johan's research group isolated and determined the structure from the subarctic sea squirt Pulmonaria genus, and synthesized pulmonarin B (Reaction Formula 1) in 3 steps with a total yield of 2.75%. Compared with several known natural acetylcholinesterase inhibitors, pulmonarin B has a stronger non-competitive acetylcholinesterase inhibitory effect (J.Nat.Prod.2014, 77, 364-369).

[0007]

[0008] In 2018, Jiang Chengshi's research group synthesized pulmonarin B (reaction formula 2) through 5 steps with a total yield of 3.69%, and reported that it has moderate acetylcholinesterase (AChE) / butyrylcholinesterase (BChE) inhibitory activity in vitro, as well as inhibition of self-induction and inhibition of acetylcholinesterase-induced β-amyloid protein aggregation, and is non-toxic to hepatocyte cell lines. The data show that pulmonarin B may become a lead compound for the development of an anti-Alzheimer's drug and a multifunctional anti-adrenaline drug (Mar. Drugs. 2018, 16, 293).

[0009]

[0010] Due to the low natural content of alkaloid pulmonarin B and the difficulty in synthesis, the research on its biological activity is not in-depth enough. The main bottleneck of the synthesis is the formation of quaternary ammonium salt in the last step. The yield of the two documents did not exceed 20%, which greatly limited the in-depth study of the biological activity of this type of compound. It is particularly important to develop efficient synthesis methods and conduct in-depth research on the agricultural activity of this type of compound. Summary of the invention

[0011] The present invention provides an efficient synthesis method of an alkaloid pulmonarin B and application of the alkaloid in preventing and treating plant viruses and pathogenic bacteria. Based on systematic literature research and reference to the synthesis method reported by the Johan research group, the present invention synthesizes pulmonarin B in 5 steps with a total yield of 59%, improves the reaction conditions, and increases the reaction yield, laying a foundation for the study of the biological activity of this type of alkaloid. The alkaloid pulmonarin B of the present invention has good anti-plant virus and anti-pathogenic activity.

[0012] The synthesis method of the alkaloid pulmonarin B of the present invention is shown in reaction formula 3: p-methoxyphenylacetic acid reacts with methanol in the presence of thionyl chloride to obtain ester 2, which is then brominated with NBS to obtain compound 3, which is hydrolyzed to obtain 4, and condensed under the catalysis of EDCI to obtain amide 5. Finally, by screening solvents and controlling the reaction time, we found that compound 5 can be converted into alkaloid pulmonarin B with a high yield of 98% by using acetone as a solvent and reacting at room temperature for 1 hour.

[0013]

[0014] The alkaloid pulmonarin B of the present invention has good anti-plant virus and pathogen activity. It can well inhibit tobacco mosaic virus (500 μg / mL inhibition rate: passivation 59%, treatment 37%, protection 43%), and the passivation activity is significantly better than the commercial variety viraviril (500 μg / mL inhibition rate: passivation 39%, treatment 37%, protection 41%). It shows a broad spectrum of inhibitory activity against 14 plant pathogens, including cucumber wilt, peanut brown spot, apple ring pattern, wheat sheath blight, corn spot, watermelon anthrax, rice seedling blight, tomato early blight, wheat fusarium, rice blast, pepper phytophthora, rape sclerotium, cucumber gray mold, and rice sheath blight.

[0015] Compared with the prior art, the present invention breaks through the synthesis barrier of the alkaloid pulmonarin B, and realizes the efficient synthesis of pulmonarin B with 5 steps and a total yield of 59%. It is the first time to find that the alkaloid pulmonarin B exhibits good anti-plant virus and anti-bacterial activity. DETAILED DESCRIPTION

[0016] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0017] Example 1: Synthesis of methyl 4-methoxyphenylacetate (2): Compound 1 (2.0 g, 11.2 mmol) was dissolved in 10 mL of methanol. In an ice-water bath, 0.5 mL of dichlorothionyl was slowly added dropwise. The reaction was carried out at 70° C. for 4 hours. The mixture was concentrated under reduced pressure to remove the solvent. 50 mL of water was added. The mixture was extracted three times with 20 mL of ethyl acetate respectively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the solvent to obtain compound 2, 2.08 g, with a yield of 95%, as an anhydrous oil. 1 H NMR (400MHz, CDCl3) δ7.23 (d, J=8.6Hz, 2H, Ar-H), 6.89 (d, J=8.6Hz, 2H, Ar-H), 3.82 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.60 (s, 2H, COCH2). 13 C NMR (100MHz, CDCl3) δ172.4, 169.5, 158.7, 155.4, 130.3, 126.1, 114.0, 55.3, 52.0, 40.3.

[0018] Example 2: Synthesis of methyl 3,5-dibromo-4-methoxyphenylacetate (3): Compound 2 (1.2 g, 6.2 mmol) was dissolved in 5 mL of acetonitrile. Under ice bath conditions, ferric chloride (1.1 g, 6.2 mmol, 1 equiv) was added. The mixture was stirred for 15 minutes. N-bromosuccinimide (NBS, 2.2 g, 12.4 mmol, 2 equiv) was slowly added. The mixture was reacted for 5 hours at room temperature. The mixture was quenched with 50 mL of water and extracted three times with 20 mL of ethyl acetate respectively. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure and desolvated. The mixture was purified by column chromatography (petroleum ether / ethyl acetate = 15 / 1), and concentrated under reduced pressure and desolvated to obtain compound 3, 1.68 g, with a yield of 75%, as a yellow oil. 1 H NMR (400MHz, CDCl3) δ7.43 (s, 2H, Ar-H), 3.87 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.54 (s, 2H, COCH2). 13 C NMR (100MHz, CDCl3) δ171.0, 153.3, 133.5, 132.4, 118.1, 60.6, 52.4, 39.6.

[0019] Example 3: Synthesis of 3,5-dibromo-4-methoxyphenylacetic acid (4): Compound 3 (0.34 g, 1.0 mmol) was dissolved in 7 mL of tetrahydrofuran and 3 mL of water, and sodium hydroxide (0.08 g, 2.0 mmol, 2 equiv) was added. The mixture was reacted at room temperature for 6 hours, and sulfuric acid solution (3 M) was added dropwise. The pH was adjusted to 1.0-2.0, and the mixture was filtered to obtain compound 4, 0.31 g, with a yield of 95%, as a white solid with a melting point of 125-130°C. 1 H NMR (400MHz, DMSO-d6) δ7.57 (s, 2H, Ar-H), 3.79 (s, 3H, OCH3), 3.60 (s, 2H, COCH2). 13 C NMR (100MHz, DMSO-d6) δ172.6, 152.5, 135.3, 134.3, 117.4, 60.8.

[0020] Example 4: Synthesis of 2-(3,5-dibromo-4-methoxyphenyl)-N-(5-dimethylaminopentyl)acetamide (5): Compound 4 (0.5 g, 1.56 mmol) was dissolved in 5 mL of dichloromethane, and N,N-dimethylpentanediamine (0.22 g, 1.72 mmol, 1.1 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.45 g, 2.34 mmol, 1.5 equiv) were added. quiv), 1-hydroxybenzotriazole (HOBT, 0.11 g, 0.78 mmol, 0.5 equiv) and triethylamine (0.48 g, 4.68 mmol, 3 equiv), under N2 protection, at room temperature, react for 6 hours, concentrate under reduced pressure to remove the solvent, add 20 mL of water, extract with 10 mL of ethyl acetate three times respectively, dry the organic layer over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent to obtain compound 5, 0.6 g, yield 89%, colorless oil. 1 H NMR (400MHz, CDCl3) δ7.45 (s, 2H, Ar-H), 5.89 (s, 1H, NH), 3.89 (s, 3H, OCH3), 3.44 (s, 2H, COCH2), 3.25 (t, J=6.1Hz , 2H, CONH-CH2), 2.27 (t, J=7.3Hz, 2H, N-CH2), 2.22 (s, 6H, N-CH3), 1.53-1.46 (m, 4H, CH2), 1.34-1.29 (m, 2H, CH2). 13 C NMR (100MHz, CDCl3) δ169.6, 153.2, 133.4, 118.3, 60.6, 59.4, 45.4, 42.1, 39.8, 29.2, 27.1, 24.6.HRMS (ESI)calcd for C 16 H 25 Br2N2O2(M+H) + 435.0277, found 435.0271.

[0021] Example 5: Synthesis of Pulmonarin B: Compound 5 (0.20 g, 0.46 mmol) was dissolved in 5 mL of acetone, iodomethane (0.10 g, 0.69 mmol, 1.5 equiv) was added, and the reaction was carried out at room temperature for 1 hour. The solution was concentrated under reduced pressure to obtain compound pulmonarin B, 0.19 g, with a yield of 98%, as an anhydrous oil. 1H NMR (400MHz, DMSO-d6) δ8.14 (s, 1H, NH), 7.53 (s, 2H, Ar-H), 3.76 (s, 3H, OCH3), 3.38 (s, 2H, COCH2), 3.27 (t, J=6.1Hz, 2H, CONH- CH2), 3.06 (t, J=7.8Hz, 2H, N-CH2), 3.06 (s, 9H, N-CH3), 1.69-1.66 (m, 2H, CH2), 1.47-1.45 (m, 2H, CH2), 1.33-1.23 (m, 2H, CH2). 13 C NMR (100MHz, DMSO-d6) δ169.6, 152.3, 136.4, 133.7, 117.5, 65.7, 60.9, 52.7, 41.0, 38.8, 29.0, 23.7, 22.2.HRMS(ESI)calcd forC 17 H 27 Br2N2O2(MI - ) + 449.0434, found 449.0437.

[0022] Example 6: Determination of anti-tobacco mosaic virus activity, the determination procedure is as follows:

[0023] 1. Virus purification and concentration determination:

[0024] Virus purification and concentration determination were carried out in accordance with the SOP specification for tobacco mosaic virus compiled by the Laboratory of the Institute of Element Analysis of Nankai University. The crude virus extract was centrifuged twice with polyethylene glycol, the concentration was determined, and the extract was refrigerated at 4°C for later use.

[0025] 2. Preparation of compound solution:

[0026] After weighing, the original drug was dissolved in DMF to obtain 1×10 5 μg / mL stock solution, and then diluted to the required concentration with an aqueous solution containing 1‰ Tween 80; Ningnanmycin preparations were directly diluted with water.

[0027] 3. In vivo protection:

[0028] Select 3-5 leaf-stage Sansi tobacco plants with uniform growth, spray the whole plant with 3 replicates per treatment, and set a 1‰ Tween 80 aqueous solution as a control. After 24 hours, sprinkle diamond dust (600 mesh) on the leaves, dip a brush in the virus solution, and gently rub the entire leaf surface along the branch vein twice, support the lower part of the leaf with the palm of your hand, the virus concentration is 10μg / mL, and rinse with running water after inoculation. Record the number of lesions 3 days later and calculate the prevention effect.

[0029] 4. In vivo therapeutic effect:

[0030] Select 3-5 leaf-stage Sansi tobacco plants with uniform growth. Use a brush to inoculate the virus on the whole leaf at a virus concentration of 10 μg / mL. Rinse with running water after inoculation. After the leaf surface is dry, spray the whole plant with the drug. Repeat 3 times for each treatment, and set a 1‰ Tween 80 aqueous solution as a control. Record the number of lesions after 3 days and calculate the prevention effect.

[0031] 5. In vivo passivation effect:

[0032] Select Sansi tobacco plants with uniform growth at the 3-5 leaf stage, mix the agent with an equal volume of virus juice and inactivate for 30 minutes, then inoculate by friction, with a virus concentration of 10 μg / mL. Rinse with running water immediately after inoculation, repeat 3 times, and set a 1‰ Tween 80 aqueous solution as a control. Count the number of lesions after 3 days and calculate the results.

[0033] Inhibition rate (%) = [(number of control necrosis spots - number of treated necrosis spots) / number of control necrosis spots] × 100%

[0034] The positive control was the commercial anti-plant virus agent ribavirin.

[0035] Table 1 Test results of anti-tobacco mosaic virus (TMV) activity of alkaloid pulmonarin B

[0036]

[0037] From the data in Table 1, it can be seen that the alkaloid pulmonarin B exhibits good anti-TMV activity, and its passivation activity is significantly better than that of ribavirin, which has great development value.

[0038] Example 7: Antibacterial activity test, the determination procedure is as follows:

[0039] In vitro bactericidal test, bacterial growth rate determination method (plate method):

[0040] Dissolve a certain amount of the drug in an appropriate amount of acetone, then dilute it to the required concentration with an aqueous solution containing 200μg / mL emulsifier, then draw 1mL of the drug solution and inject it into the culture dish, then add 9mL of culture medium respectively, shake well to make a 50μg / mL drug-containing plate, and use the plate with 1mL of sterilized water as a blank control. Use a 4mm diameter puncher to cut the bacterial disc along the outer edge of the hyphae and move it to the drug-containing plate. Repeat each treatment three times. Place the culture dish in a constant temperature incubator at 24±1℃ for culture. After 48 hours, investigate the expansion diameter of the bacterial disc of each treatment, calculate the average value, and calculate the relative inhibition rate compared with the blank control.

[0041]

[0042] Table 2 In vitro bactericidal activity test results of alkaloid pulmonarin B:

[0043]

[0044] The alkaloid pulmonarin B showed broad-spectrum inhibitory activity against 14 tested bacteria at a test concentration of 50 μg / mL.

[0045] The raw materials and reagents involved in the above examples are all commercially available or prepared by reference methods, and the chemical reaction processes are within the skill of those skilled in the art.

[0046] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. The use of alkaloid pulmonarin B in preventing and treating plant viral diseases, characterized in that: The plant virus is tobacco mosaic virus.

2. The use of alkaloid pulmonarin B in preventing and treating plant pathogenic diseases, characterized in that: The pathogens are 14 plant pathogens including cucumber wilt, peanut brown spot, apple ring pattern, wheat leaf blight, corn leaf spot, watermelon anthrax, rice seedling blight, tomato early blight, wheat fusarium, rice blast, pepper phytophthora, rapeseed sclerotium, cucumber gray mold and rice leaf blight.

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